Size enhanced hemoglobins: surface decoration and crosslinking of the protein with polyoxy alkylene glycols

ABSTRACT

Novel modified hemoglobins comprising polyalkylene glycols and novel methods for making those hemoglobins are provided. One group of modified hemoglobins comprise polyalkylene glycols bonded to the hemoglobin with an amide linkage at Glu-43(β). Additional polyalkylene glycols can also be bonded to the Glu-22(β) and/or the Asp-47(β). These hemoglobins are made by a novel amidation procedure. A second group of modified hemoglobins comprise a polyalkylene glycol covalently bonded to the hemoglobin at the α-amino of a Val-1(β) or a Val-1(α). Additional polyalkylene glycols can optionally be covalently bonded to a limited number of ε-amino groups. This second group of hemoglobins is made using a novel reductive alkylation procedure. A third group of modified hemoglobins comprise a polyalkylene glycol bonded to a thiol group of the hemoglobin through a phenylsuccinimido linkage, wherein no polyalkylene glycol is bonded to a Cys-93(β). This third group of modified hemoglobins is made by an improvement in a hemoglobin-polyalkylene linkage procedure utilizing thiolation-mediated maleimide chemistry.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/306,623, filed Jul. 19, 2001.

BACKGROUND

(1) Field of the Invention

The present invention generally relates to novel modified hemoglobin andnovel methods for modifying hemoglobin. More specifically, the inventionrelates to novel hemoglobin compositions comprising polyalkylene glycolsand methods for making those hemoglobin compositions.

(2) Description of the Related Art

References cited:

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Hemoglobin (Hb) is the major constituent of the erythrocyte whichcarries oxygen from the lungs throughout the body. When contained in redblood cells, Hb exists as a tetramer structure composed of two oxygenlinked αβ dimers, each having a molecular weight of about 32 Kd. Each αand β subunit of each dimer has a protein chain and a heme molecule.

The sequences of the α and β protein chains are known. Hb is apotentially useful blood substitute for transfusions, and has beenproposed as a reagent to trap nitric oxide in septic shocks, and tomodulate tissue oxygenation during radiation therapy of cancer.Recombinant DNA technology also has afforded the generation of modifiedHb with oxygen affinities modulated for special needs of individualtherapeutic applications.

The potential use of Hb as blood substitutes in transfusions or othertherapeutic applications, however, has been hampered by the shortcirculation half-life of Hb. In solution outside of the red blood cell,Hb readily dissociates from its tetrameric form into its dimers and evenmonomers, which are rapidly filtered through the kidneys. Accordingly, amultitude of methods for crossbridging Hb (e.g. bifunctionalmodification) and other means for increasing the hydrodynamic volume ofHb (e.g. monofunctional decoration) have been devised to limit orprevent the extravasation of Hb.

Simon and Konigsberg (1966) reports the use of bis(N-maleimidomethyl)ether (BME) to generate intramolecularly crosslinked Hb. Bunn et al.(1969) later reported that BME crosslinked Hb increased the half-life ofHb four-fold when infused into rats and dogs. However, the crosslinkingof Hb with BME resulted in a concomitant increase in the oxygen affinityof Hb which prevented its use as a potential Hb-based oxygen carrier.

Xue and Wong (1994) describes many of the current methods forcrosslinking Hb. These include the use of dextran, hydroxyethyl starch,inulin, polyvinylpyrrolidone, and polyethylene glycol as crosslinkersfor Hb. Other crosslinkers include glycoaldehyde and glutaraldehyde(MacDonald and Pepper, 1994); bis(3,5-dibromosalicyl) fumarate (Walder,R. Y., et al., 1994); acyl phosphate esters (Kluger et al., 1994 U.S.Pat. No. 5,334,707); bissulfosuccinimidyl esters of aliphaticdicarboxylic acids (Manjula et al., 1994); and benzenepentacarboxylate(U.S. Pat. No. 5,349,054).

Nho et al. (1994) describes the monofunctional decoration of hemoglobinwith polyethylene glycol. Similar modification of the hemoglobinmolecule are described in U.S. Pat. Nos. 4,301,144; 4,412,989;4,670,417; 5,234,903; and 5,312,808, and WIPO publication WO 94/04193.

The various modified hemoglobins described in the references cited abovecan be broadly grouped into three classes. (i) intramolecularlycrossbridged Hb tetramers, (ii) inter and intra molecularly crossbridged Hb polymers, and (iii) Hb surface decorated with inert polymerssuch as polyethylene glycol (PEG). All these three classes of modifiedhemoglobins prevent glomerular filtration of acellular Hb and hence donot exhibit any nephrotoxicity that is associated with unmodifiedacellular Hb. However, the intrinsic propensity of Hb to bind nitricoxide, and hence its ability to influence vascular tone when present asan acellular component has been an obstacle to the widespread adoptionof acellular Hb for therapeutic purposes. The different classes ofmodified Hb exhibit different degrees of vasoactivity. Theintramolecularly crossbridged Hb, with its molecular size of 64,000daltons, exhibited the highest vasoactivity, which is comparable to thatof unmodified Hb. The intra and intermolecularly crossbridged species ofHb, with apparent molecular size of 200,000 to 300,000 exhibit asomewhat lowered vasoactivity relative to the parent Hb (orintramolecularly crossbridged HbA). The samples of Hb surface decoratedwith PEG chains with an apparent molecular size of 275,000 daltons orhigher do not exhibit any vasoactivity. Thus, the increased molecularsize of Hb appears to have reduced the vasoactivity of the product,presumably minimizing the extravasation of the sample into theinterstitial space. This observation has presented a new approach toovercome the vasoactivity mediated toxicity of acellular Hb, distinctfrom approaches under development to engineer the Hb molecule throughsite directed mutagenesis to suppress the affinity of heme towardsnitric oxide. The higher viscosity and the colloidal osmotic pressure ofthe solutions of surface decorated Hb appears to have other beneficialeffects as well (Vandegriff et al., 1997; Winslow et al., 1998).

The surface decorated Hb investigated to date carry about ten PEG-5000chains per tetramer (total mass of about 50,000 daltons of PEG pertetramer). The PEG-chains in this sample are linked to the surface αand/or ε-amino groups of Hb through isopeptide linkage (succinimidylchemistry based PEGalation). Such preparations of surface decorated Hbdo not increase the blood pressure, systemic vascular resistanceremained unchanged, and tissue oxygenation are maintained at a levelcomparable to that of blood, even though the oxygen affinity of thesepreparations are higher than that of erythrocytes (Winslow et al.,1998).

One of the limitations of the above-described succinimidyl chemistry forsurface decoration of Hb with PEG chains at the amino groups of Hb, isthat the isopeptide linkage generated between Hb and the PEG-moleculedoes not carry the original positive charge of the amino (α or ε) of Hb.To overcome this limitation of earlier surface decoration chemistry, anovel protocol was recently developed to attach PEG-chains to Hb usingthe ε-amino groups of its surface Lys residues wherein the Hb stillretains the original positive charge of the amino groups (U.S. Pat. No.5,585,484). This involves amidination of the ε-amino groups of Hb byiminothiolane to introduce sulfhydryl groups on to the protein, whichare subsequently targeted as the modification sites for PEGalation usingmaleimide chemistry-based PEG reagents. This approach has at least twoadditional specific advantages over the previously used succinimidylchemistry: (1) the very high reactivity and selectivity of the maleimidebased reagents to the sulfhydryl groups facilitates the nearquantitative modification of the thiols with a limited excess of thereagents (in this case maleidophenyl PEG-chains), and (2) the thiolgroup of iminothiolane is latent and is generated only in situ as aconsequence of the reaction of the reagent with the protein aminogroups. Accordingly, Hb can be incubated simultaneously with thethiolating and the PEGalating reagents for surface decoration withPEG-chains.

Despite its advantages, the thiolation mediated, maleimide chemistrybased surface decoration procedure described in U.S. Pat. No. 5,585,484results in the modification of Cys-93(β) by the maleidophenyl PEG. Thismodification leads to an increase in the oxygen affinity of Hb.

Most of the known compounds used to modify Hb are difficult tosynthesize, do not modify Hb in an efficient manner, cannot bemanipulated quantitatively to form the desired modification, and/orlower or raise the oxygen affinity of the modified hemoglobin.Accordingly, there exists a need for new synthetic compounds, and newmethods which can modify hemoglobin in an efficient and focused manner,and do not substantially affect the oxygen affinity of the modifiedhemoglobin. The present invention satisfies this need.

SUMMARY OF THE INVENTION

Accordingly, the inventors have succeeded in developing three novelmethods for creating novel modified hemoglobins comprising polyalkyleneglycols. The hemoglobins provide certain properties that are superior toother modified hemoglobins.

Thus, one embodiment of the present invention is directed to ahemoglobin comprising a polyalkylene glycol, wherein the polyalkyleneglycol is a polypropylene glycol or a polyethylene glycol (PEG), and thepolyalkylene glycol is covalently bonded to the hemoglobin with an amidelinkage at a Glu-43(β). Preferably, the polyalkylene glycol is a PEG,and the hemoglobin is a hemoglobin A. In other preferred embodiments,the polyalkylene glycol is a PEG, and the hemoglobin further comprises asecond PEG wherein the second PEG is covalently bonded to a Glu-22(β)with an amide linkage. In some aspects of these embodiments, thepolyalkylene glycol is a PEG, and the PEG does not crosslink thehemoglobin intramolecularly or intermolecularly. Preferably, thesehemoglobins have at least 6 PEGs bonded to the hemoglobin through anamide linkage. In other aspects of these embodiments, the polyalkyleneglycol is a PEG, and the PEG intramolecularly crosslinks the hemoglobin,or intermolecularly crosslinks the hemoglobin with a second hemoglobin.

Other embodiments of the invention are directed to a hemoglobincomposition comprising hemoglobin (Hb) decorated with one or more PEGmolecules. In these embodiments, the Hb-PEG has the formulaHb-(CO—NH—CHR—CO—W—CH₂—CH₂—[O—CH₂—CH₂]_(n)—R′)_(m) wherein n is aninteger from about 125 to about 500, m is an integer from 1 to 10, W isNH or 0, R is an amino acid side chain, R′ is selected from the groupconsisting of OH, OCH₃, CH₂OH, CH₂OCH₃, CH₂CH₂OH, and CH₂CH₂OCH₃, andwherein at least one PEG is bonded to the Hb at Glu-43(β). Preferably,the Hb is a hemoglobin A, W is NH, n is about 125, m is 6-8, R is H orCH₂COOH, and R′ is CH₂CH₂OCH₃.

Additional embodiments provide a hemoglobin composition comprising atleast one hemoglobin molecule (Hb), crosslinked by one or more PEGmolecules, wherein the crosslinked Hb has the formulaHb-CO—NH—CHR—CO—W—CH₂—CH₂—[O—CH₂—CH₂]_(n)—NH—CO—W′—CHR—NH—CO-Hb′ whereinHb and Hb′ are the same or different hemoglobin molecule, n is aninteger from about 15 to about 250, W and W′ are each independently NHor O, R is an amino acid side chain, and HbA and/or HbA′ is bonded tothe PEG with an amide linkage at Glu-43(β). Preferably, R is H orCH₂COOH, Hb and Hb′ are different hemoglobin A tetramers, W and W′ areboth NH, and the PEG intermolecularly crosslinks HbA with HbA′.

In additional embodiments, the present invention is directed to a methodof producing a hemoglobin comprising a polyalkylene glycol, wherein thepolyalkylene glycol is polypropylene glycol or polyethylene glycol(PEG). The method comprises mixing in a suitable buffer (a) thehemoglobin, (b) a carbodiimide, and (c) a nucleophilic polyalkyleneglycol with a terminal amine having a pK_(a) below 9, and incubating themixture under conditions and for a time sufficient for the polyalkyleneglycol to covalently bond to the hemoglobin at Glu-43(β). In preferredaspects of these embodiments, the polyalkylene glycol is a PEG, and 6 to8 PEG molecules bind to the hemoglobin. In other preferred aspects, thehemoglobin is hemoglobin A, the buffer is MES buffer at pH 6-8, thecarbodiimide is 1-ethyl-3-(3′-dimethyiaminopropyl)carbodiimide, and themixture further comprises N-hydroxysulfosuccinimide. Preferably, thecarbodiimide is present in the mixture at about 10-50 mM.

In still other embodiments, the present invention is directed to amethod of producing a hemoglobin comprising a polyethylene glycol (PEG).The method comprises mixing in a suitable buffer (a) the hemoglobin, (b)a carbodiimide, and (c) a nucleophilic PEG with a terminal amine havinga pK_(a) below 9, and incubating the mixture under conditions and for atime sufficient for the PEG to covalently bond to the hemoglobin atGlu-43(β). In these embodiments, the nucleophilic PEG has the formulaH₂N—CHR—CO—W—CH₂—CH₂—[O—CH₂—CH₂]_(n)—R′ wherein n is an integer fromabout 125 to about 500, W is NH or O, R is an amino acid side chain, andR′ is selected from the group consisting of OH, OCH₃, CH₂OH, CH₂OCH₃,CH₂CH₂OH, and CH₂CH₂OCH₃; preferably, the hemoglobin is a hemoglobin A,W is NH, n is about 125, R is H or CH₂COOH, and R′ is CH₂CH₂OCH₃.

In other aspects of these methods, the nucleophilic PEG has the formulaH₂N—CHR—CO—W—CH₂—CH₂—[O—CH₂—CH₂]_(n)—NH—CO—W′—CHR′—NH₂ wherein n is aninteger from about 15 to about 250, W and W′ are each independently NHor O, and R and R′ are each independently an amino acid side chain;preferably W and W′ are both NH, and R or R′ is each independently H orCH₂COOH.

The present invention is also directed to a hemoglobin comprising apolyalkylene glycol, wherein the polyalkylene glycol is polypropyleneglycol or polyethylene glycol (PEG), and wherein the polyalkylene glycolis covalently bonded to the hemoglobin at the α-amino of a Val-1(β).Preferably, the polyalkylene glycol is a PEG, and the hemoglobin ishemoglobin A. In other preferred embodiments, the hemoglobin furthercomprises a second PEG wherein the second PEG is covalently bonded tothe hemoglobin at the α-amino of a Val-1(α). In some aspects of theseembodiments, the polyalkylene glycol does not crosslink the hemoglobinintramolecularly or intermolecularly; in other aspects the polyalkyleneglycol does crosslink the hemoglobin. In preferred embodiments, at least4 polyalkylene glycols are bonded to the hemoglobin at an amino moiety.

In preferred embodiments, the invention is directed to a hemoglobincomposition comprising hemoglobin A (HbA) decorated with one or more PEGmolecules, wherein the HbA-PEG has the formulaHbA-(NH—CH—[CH₂]_(p)—[NH]_(q)—CH₂—CH₂—[O—CH₂—CH₂]_(n)—R)_(m) wherein pis an integer from 2 to 6, q is 1 or 2, n is an integer from about 125to about 500, m is an integer from 1 to 10, and R is selected from thegroup consisting of OH, OCH₃, CH₂OH, CH₂OCH₃, CH₂CH₂OH, and CH₂CH₂OCH₃.Preferably, q is 1, n is about 125, m is 1-4, and R is CH₂CH₂OCH₃.

In other preferred embodiments the invention is directed to a hemoglobincomposition comprising a hemoglobin (Hb) crosslinked by one or more PEGmolecules, wherein the crosslinked Hb has the formulaHb-NH—CH—[CH₂]_(p)—[NH]_(q)—CH₂—CH₂—[O—CH₂—CH₂]_(n)—[NH]_(q)—[CH₂]_(p)—CH—NH-Hb′wherein Hb and Hb′ are the same or different hemoglobin molecule, each pis independently an integer from 2 to 6, each q is 1 or 2, and n is aninteger from about 15 to about 250. Preferably, q is 1. Hb and Hb′ canbe the same or different hemoglobin A tetramer.

The invention is also directed to a method of producing a hemoglobincomprising a polyalkylene glycol, wherein the polyalkylene glycol is apolypropylene glycol or a polyethylene glycol (PEG), and wherein the PEGis covalently bonded to the hemoglobin at the α-amino of a Val-1(β). Themethod comprises incubating the hemoglobin with a polyalkylene glycolaldehyde and a borohydride under conditions and for a time sufficientfor the polyalkylene glycol aldehyde to bond to the hemoglobin at theVal-1(β). In preferred embodiments of these methods, the polyalkyleneglycol is a PEG, the Hb is hemoglobin A, and the borohydride is sodiumcyano borohydride. Preferably, a second PEG aldehyde also bonds to thehemoglobin at a Val-1(α). In particularly preferred embodiments, atleast 4 PEG aldehydes bond to the hemoglobin at free amino groups. Insome embodiments, the polyalkylene glycol does not crosslink thehemoglobin intramolecularly or intermolecularly; in other embodiments,the polyalkylene glycol does crosslink the hemoglobin.

In additional embodiments, the present invention is directed to a methodof producing a hemoglobin comprising a polyethylene glycol (PEG),wherein the PEG is covalently bonded to the hemoglobin at the α-amino ofa Val-1(β). The method comprises incubating the hemoglobin with a PEGaldehyde and a borohydride under conditions and for a time sufficientfor the PEG to bond to the hemoglobin at the Val-1(β), wherein the PEGaldehyde has the formulaHOC-[CH₂]_(p)—[NH]_(q)—CH₂—CH₂—[O—CH₂—CH₂]_(n)—R wherein p is an integerfrom 2 to 6, q is 1 or 2, n is an integer from about 125 to about 500,and R is selected from the group consisting of OH, OCH₃, CH₂OH, CH₂OCH₃,CH₂CH₂OH, and CH₂CH₂OCH₃. Preferably, q is 1, n is about 125, and R isCH₂CH₂OCH₃.

In still other embodiments, the present invention is directed to amethod of producing a hemoglobin comprising a polyethylene glycol (PEG),wherein the PEG is covalently bonded to the hemoglobin at the α-amino ofa Val-1(β) and crosslinks the hemoglobin intramolecularly orintermolecularly. The method comprises incubating the hemoglobin with aPEG aldehyde and a borohydride under conditions and for a timesufficient for the PEG to bond to the hemoglobin at the Val-1(β),wherein the PEG aldehyde has the formulaOHC—[CH₂]_(p)—[NH]_(q)—CH₂—CH₂—[O—CH₂—CH₂]_(n)—[NH]_(q)—[CH₂]_(p)—CHOwherein each p is independently an integer from 2 to 6, each q isindependently 1 or 2, and n is an integer from about 15 to about 250.

The present invention is additionally directed to a hemoglobincomprising a polyalkylene glycol, wherein the polyalkylene glycol is apolypropylene glycol or a polyethylene glycol (PEG), wherein thepolyalkylene glycol is covalently bonded to the hemoglobin at a thiolmoiety through a phenylsuccinimido linkage, and wherein no polyalkyleneglycol is covalently bonded to a Cys-93(β). Preferably, the polyalkyleneglycol is a PEG, and the hemoglobin is hemoglobin A. In theseembodiments, the polyalkylene glycol can crosslink the hemoglobin or candecorate the hemoglobin.

In still other embodiments, the invention is directed to a hemoglobincomprising a polyethylene glycol (PEG), wherein the PEG is covalentlybonded to the hemoglobin at a thiol moiety through a phenylsuccinimidolinkage, and wherein no PEG is covalently bonded to a Cys-93(β). Thehemoglobin has the formula Hb-(S—Y—R—CH₂—CH₂—[O—CH₂—CH₂]_(n)—R′—Y′)_(m)wherein n is an integer from about 125 to about 500; m is an integerfrom about 2 to about 16; R is carbamate, urea, or amide; R′ iscarbamate, urea, amide, or oxygen; Y is 4-phenylsuccinimido or3-phenylsuccinimido; and Y′ is methyl or hydrogen. Preferably, the Hb ishemoglobin A, Y is 4-phenylsuccinimido, Y′ is methyl, R is carbamate, R′is oxygen, n is about 125, and M is an integer from about 6 to about 8.

The invention is also directed to a hemoglobin comprising a polyethyleneglycol (PEG), wherein the PEG is covalently bonded to the hemoglobin ata thiol moiety through a phenylsuccinimido linkage, and wherein no PEGis covalently bonded to a Cys-93(β), the hemoglobin having the formulaHb-(S—Y—R—CH₂—CH₂—[O—CH₂—CH₂]_(n)—R′—Y′—S-Hb′ wherein n is an integerfrom about 15 to about 250, R and R′ are the same or different and arecarbamate, urea, or amide, Y and Y′ are the same or different and are4-phenylsuccinimido or 3-phenylsuccinimido, and Hb and Hb′ are the sameor different hemoglobin molecule. Preferably, Y and Y′ are both4-phenylsuccinimido, and R and R′ are both carbamate.

In still other embodiments, the invention is directed to a method formaking a hemoglobin comprising a polyalkylene glycol, wherein thepolyalkylene glycol is polypropylene glycol or polyethylene glycol(PEG), and wherein the polyalkylene glycol is covalently bonded to thehemoglobin at a thiol moiety through a phenylsuccinimido linkage. Themethod comprises (a) conjugating a reagent to a Cys-93(β) sulfhydrylgroup of the hemoglobin by a reversible method; (b) treating thehemoglobin with a maleidophenyl polyalkylene glycol and iminothiolaneunder conditions and for a time sufficient for the polyalkylene glycolto conjugate to the thiol moiety of the hemoglobin through aphenylsuccinimido linkage; and (c) removing the reagent from theCys-93(β) sulfhydryl group of the hemoglobin. In preferred embodiments,the polyalkylene glycol is a PEG. The reagent can be an oxidizedglutathione or, preferably, a dithiopyridine or a mixed disulfide ofpyridine and polyalkylene glycol. A preferred mixed disulfide ofpyridine and polyalkylene glycol is a mixed disulfide of pyridine andPEG-5000.

The present invention is additionally directed to a method for making ahemoglobin comprising a polyethylene glycol (PEG), wherein the PEG iscovalently bonded to the hemoglobin at a thiol moiety through aphenylsuccinimido linkage. The method comprises (a) conjugating areagent to a Cys-93(β) sulfhydryl group of the hemoglobin by areversible method; (b) treating the hemoglobin with a maleidophenyl PEGand iminothiolane under conditions and for a time sufficient for the PEGto conjugate to the thiol moiety of the hemoglobin through aphenylsuccinimido linkage; and (c) removing the reagent from theCys-93(β) sulfhydryl group of the hemoglobin, wherein the hemoglobincomprising the PEG has the formulaHb-(S—Y—R—CH₂—CH₂—[O—CH₂—CH₂]_(n)—R′—Y′)_(m) wherein n is an integerfrom about 125 to about 500; m is an integer from about 2 to about 16; Ris carbamate, urea, or amide; R′ is carbamate, urea, amide, or oxygen; Yis 4-phenylsuccinimido or 3-phenylsuccinimido; and Y′ is methyl orhydrogen. In preferred embodiments, the Hb is hemoglobin A, Y is4-phenylsuccinimido, Y′ is methyl, R is carbamate, R′ is oxygen, n isabout 200, and M is an integer from about 6 to about 8.

In still other embodiments, the present invention is directed to amethod for making a hemoglobin comprising a polyethylene glycol (PEG),wherein the PEG is covalently bonded to the hemoglobin at a thiol moietythrough a phenylsuccinimido linkage. The method comprises (a)conjugating a reagent to a Cys-93(β) sulfhydryl group of the hemoglobinby a reversible method; (b) treating the hemoglobin with a maleidophenylPEG and iminothiolane under conditions and for a time sufficient for thePEG to conjugate to the thiol group of the hemoglobin through aphenylsuccinimido linkage; and (c) removing the reagent from theCys-93(β) sulfhydryl group of the hemoglobin. The hemoglobin comprisingthe PEG has the formula Hb-(S—Y—R—CH₂—CH₂—[O—CH₂—CH₂]_(n)—R′—Y′—S-Hb′wherein n is an integer from about 15 to about 250, R and R′ are thesame or different and are carbamate, urea, or amide, Y and Y′ are thesame or different and are 4-phenylsuccinimido or 3-phenylsuccinimido,and Hb and Hb′ are the same or different hemoglobin molecule.Preferably, Y and Y′ are both 4-phenylsuccinimido, and R and R′ are bothcarbamate. Hb and Hb′ can be the same or different hemoglobin Atetramers.

The invention is additionally directed to an improved PEGalatedhemoglobin comprising a PEG covalently bonded to the hemoglobin at athiol moiety through a phenylsuccinimido linkage. In these embodiments,the improvement comprises an absence of a PEG bonded to a Cys-93(β).Preferably, the phenylsuccinimido linkage is a 4-phenylsuccinimidolinkage.

In additional embodiments, the invention is directed to an improvementin a method for producing a PEGalated hemoglobin comprising PEGcovalently bonded to the hemoglobin at a thiol moiety through aphenylsuccinimido linkage, where the method comprises treating thehemoglobin with a maleidophenyl PEG and iminothiolane under conditionsand for a time sufficient for the PEG to conjugate to the thiol moietyof the hemoglobin through a phenylsuccinimido linkage. The improvementcomprises conjugating a reagent to a Cys-93(β) sulfhydryl group of thehemoglobin by a reversible method before treating the hemoglobin withthe PEG, then removing the reagent after treating the hemoglobin withthe PEG. The reagent can be oxidized glutathione or, preferably,dithiopyridine or a mixed disulfide of pyridine and a polyalkleneglycol. A preferred mixed disulfide of a pyridine and a polyalkyleneglycol is a mixed disulfide of pyridine and PEG-5000.

The present invention is also directed to a hemoglobin comprising a PEG,produced by any of the methods described above. Additionally, any of thehemoglobin compositions provided above can be usefully provided in apharmaceutically acceptable excipient, for use in therapeuticapplications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the amidation of carboxyl groupsof HbA in the presence of EDC, Sulfo-NHS, and glycinamidyl PEG-5000.

FIG. 2 is the structure of the amino acid ester of PEG.

FIG. 3 is a graph representing the elution profile of size exclusionchromatography of HbA amidated with the glycine ester of PEG-5000 in thepresent of 10 mM EDC for 1 hour at pH 7.0 and room temperature. Afterthe reaction, the sample was first gel filtered on a G-25 column toremove the excess reagents. The peak marked 64 K represents HbA, and thepeak migrating to the 128 K marker represents HbA PEGalated atGlu-43(β).

FIG. 4 is a graph representing the elution profile ofXL-fumaryl-Lys-99-αα HbA intermolecularly crosslinked withbis-maleidophenyl PEG-600. Each new component eluting earlier thancrosslinked HbA present in the crosslinked sample represents the elutionchanges associated with an increase in the molecular size correspondingto 64,000 daltons.

FIG. 5 is a graph showing the correlation between the apparent increasein the molecular size of HbA (as reflected by size exclusionchromatography) resulting from its amidation with the glycine ester ofPEG-5000. HbA was amidated in the presence of 10, 25 and 50 mM EDC todetermine the elution positions for HbA containing two, four and sixcopies of PEG-5000 chains per tetramer.

FIG. 6 is a schematic representation of reductive alkylation of aminogroups of Hb using PEG-aldehyde.

FIG. 7 shows the structure of PEG-propionaldehyde for surface decorationof HbA targeted to α-amino groups of HbA.

FIG. 8 is a graph representing the elution profile of HbA reacted withPEG-20,000 maleimide on Superose®-12 columns. The elution position ofsamples of HbA decorated with PEG-5000 is indicated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed toward novel hemoglobin (Hb)compositions, and novel methods of preparing those compositions. The Hbcompositions comprise hemoglobin molecules that are covalently bonded topolyalkylene (=polyoxy alkylene) glycols by methods that retain thefunctionality of the hemoglobin's Cys-93(β) residues, which areimportant for maintaining a low oxygen affinity.

Three novel chemical methods are described for covalently bonding thepolyalkylene glycol to the Hb, while avoiding the Cys-93(β) residues.All three methods are directed to the production of Hbs bonded topolyalkylene glycols, where the bonding is at precise amino acidresidues of the Hb. Additionally, all three methods can be adjusted toallow increasing or decreasing amounts of polyalkylene glycols to bebonded to the Hb, thus allowing the production of Hbs with as few as oneor as many as 10 or more polyalkylene glycols attached. The first methodemploys carbodiimide to form an amide linkage with Hb carboxyl moieties;the second utilizes reductive alkylation chemistry to bond thepolyalkylene glycol to amino residues of the Hb; and the third is animprovement of the previously developed thiolation mediated maleimidemethod that bonds the polyalkylene glycol to an Hb thiol moiety throughphenylsuccinimido linkage, wherein the improvement avoids polyalkyleneglycol bonding to the Cys-93(β) thiol moiety.

Hemoglobin, or “Hb” is defined herein as (a) an iron-containingrespiratory pigment found in vertebrate red blood cells that consists ofglobin composed of four subunits (a tetramer) each of which is linked toa heme molecule, that functions in oxygen transport to the tissues afterconversion to oxygenated form in the gills or lungs, and that assists incarbon dioxide transport back to the gills or lungs after surrender ofits oxygen; and (b) recombinantly produced hemoglobin; ap-dimers ofhemoglobin, inter- or intramolecularly crosslinked hemoglobin, as wellas modified versions of the hemoglobins provided above, which includebut are not limited to modifications increasing or decreasing the oxygenaffinity of hemoglobin. All hemoglobins of the present invention arecapable of binding heme and are comprised of at least an αβ-dimer. Thehemoglobins of the present invention are preferably from mammals, andmore preferably from human beings. In the most preferred embodiments,the hemoglobin is hemoglobin A (HbA).

As used herein, a polyalkylene glycol includes any compound of theformula X—[O—R]_(n)—Y, wherein R is an alkyl group, n is an integer fromabout 5 to about 1000, and X and Y can be anything. However, when thepolyalkylene glycol is used in the methods of the present invention toproduce the novel Hb-polyalkylene glycol compositions described herein,at least one of X or Y must comprise a functional group that is requiredin the method. For the amidation method, that functional group is anamino group; for the reductive alkylation method the functional group isan aldehyde; and for the improved thiolation mediated maleimide method,the functional group is 3- or 4-phenylmaleimido. The skilled artisancould, without undue experimentation, determine numerous X or Yconstituents that would be useful for various applications of thepresent invention.

In preferred embodiments, R is CH₂—CH₂, wherein the polyalkylene glycolis a polyethylene glycol (PEG), or R is CH₂—CH₂—CH₂, wherein thepolyalkylene glycol is polypropylene glycol. These polyalkylene glycolsare preferred because they are readily commercially available. In themost preferred embodiments, the polyalkylene glycol is PEG, because PEGis commercially available in many forms (see, e.g., ShearwaterCorporation (Huntsville, Ala.) catalog, at www.shearwatercorp.com. ThePEGs or polypropylene glycols useful for the present invention do notneed to have R groups that are completely CH₂—CH₂ or CH₂—CH₂—CH₂,respectively. The skilled artisan would understand that PEGs orpolypropylene glycols useful for the present invention could have asubstantial amount of substitutions in these R groups. Therefore,encompassed within the definition of PEG or polypropylene glycol areforms wherein the R groups are a majority of CH₂—CH₂ or CH₂—CH₂—CH₂,respectively, and wherein the Hb-PEG or Hb-polypropylene glycol madeusing that PEG or polypropylene glycol according to the inventionretains the same oxygen affinity and vasoactivity as the analogousHb-PEG or Hb-polypropylene glycol having R groups that are exclusivelyCH₂—CH₂ or CH₂—CH₂—CH₂, respectively.

The methods provided herein can be used for producing novelHb-polyalkylene glycol compositions wherein the polyalkylene glycoldecorates the Hb, as well as wherein the polyalkylene glycol crosslinksthe Hb. As used herein, the term “decorate” or “surface decorate” refersto the characteristic of the Hb-polyalkylene glycol wherein thepolyalkylene glycol is bonded to the Hb at only one point, usually atone end of the polyalkylene glycol, such that the polyalkylene glycoldoes not crosslink the Hb with itself or another Hb. By contrast, theterm “crosslink” refers to the characteristic of the Hb-polyalkyleneglycol wherein the polyalkylene glycol is bonded to an Hb at two points,usually at each end, such that the polyalkylene glycol links togethertwo subunits of the same Hb (“intramolecular crosslink”) or thepolyalkylene glycol links together different Hb molecules(“intermolecular crosslink”). When more than one polyalkylene glycol isbonded to a Hb, there can be both intra- and intermolecularcrosslinking.

As is generally known in the art, polyalkylene glycols of various sizescan be utilized to decorate or crosslink Hb. For decoration, preferredsizes are polyalkylene glycols of 5,000-20,000 daltons, which isequivalent to about 125 to about 500 —O—CH₂—CH₂ units when thepolyalkylene glycol is PEG, although other sizes can be useful. Forintramolecular crosslinking, polyalkylene glycols of 600-5000 daltons(equivalent to a PEG with about 15-125 —O—CH₂—CH₂— units) areparticularly useful; for intermolecular crosslinking, polyalkyleneglycols of 600-10,000 daltons (equivalent to a PEG with about 15-250—O—CH₂—CH₂— units) are particularly useful.

As discussed in Example 4, the inventors have further discovered anunexpected useful property of the polyalkylene glycol-modifiedhemoglobins of the present invention, particularly those where thepolyalkylene glycol is used to surface decorate the hemoglobin. Theinventors have discovered that the modified hemoglobins have anincreased hydrodynamic volume that has an apparent molecular weight,when measured by molecular sieve chromatography, that is much greaterthan its true molecular weight. Where the polyalkylene glycol is used tosurface decorate the hemoglobin, the increased volume reflects a size ofhemoglobin that is nearly six times the molecular mass of thepolyalrylene glycol that is bonded to it. This increased hydrodynamicsize is known to be advantageous.

I. Production of Hb-polyalkylene Glycols by Amidation.

The inventors disclose herein the discovery that various nucleophilicpolyalkylene glycol molecules that have a free terminal amine with apK_(a) below about 9.0 can be bonded to basic carboxyl moieties of Hbthrough modifications of an amidation procedure that has been previouslydeveloped (Rao and Acharya, 1994). However, because the previouslydisclosed amidation procedure was developed for attaching only verysmall molecules to Hb, the skilled artisan would understand that,without the instant disclosure, the amidation procedure would notnecessarily be useful for attaching the relatively large polyalkyleneglycol molecules to Hb. The methods comprise mixing, in a suitablebuffer, the hemoglobin, the polyalkylene glycol and a carbodiimide. Inpreferred embodiments, the carbodiimide is1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDC), and the mixturealso comprises N-hydroxysulfosuccinimide (FIG. 1). TheN-hydroxysulfosuccinimide increases the efficiency of the reaction by“trapping” the carbodiimide-activated carboxyl group of the Hb (Id.).

In these methods, if a relatively low carbodiimide concentration isutilized in the mixture (i.e., about 10 mM), the methods result inbonding of the polyalkylene glycol at the Hb Glu-43(β) residues. Withincreasing carbodiimide concentrations (i.e., up to 50 mM or more),bonding also occurs at the Glu-22(β) position, then at the Asp-47(β)position. Bonding can also occur at the terminal carboxyl position (theα-carboxyl groups of Arg-141(α) and/or His-146(β)), or at other carboxylresidues, albeit with greater difficulty, requiring higher carbodiimideconcentrations, a higher ratio of polyalkylene glycol:Hb, and/or alonger incubation time. Thus, by varying the reaction conditions usingroutine experimentation, varying amounts of the polyalkylene glycol canbe attached to the Hb, up to 10 or more polyalkylene glycols on an HbAtetramer. In preferred embodiments where the polyalkylene glycol is usedfor decorating the hemoglobin, the resulting Hb is an HbA with about 6-8polyalkylene glycols, wherein the polyalkylene glycols are PEG-5000(having about 125 —O—CH₂—CH₂ units).

The skilled artisan could identify, without undue experimentation,numerous nucleophilic polyalkylene glycols having a terminal amine witha pK_(a) below 9 that are useful for the present invention. Usefulexamples are amino acid esters of PEG (FIG. 2), particularly where R═H(glycine ester) or CH₂—COO— (aspartic acid ester). For decoration, thenucleophilic polyalkylene glycol should have only one reactive terminalamine; for crosslinking, there should be two terminal amines, preferablyat each end of the polyalklene glycol molecule. In preferredembodiments, the polyalkylene glycol for decoration is a PEG with theformula H₂N—CHR—CO—W—CH₂—CH₂—[O—CH₂—CH₂]_(n)—R′ wherein n is an integerfrom about 125 to about 500, W is NH or O, R is an amino acid sidechain, and R′ is selected from the group consisting of OH, OCH₃, CH₂OH,CH₂OCH₃, CH₂CH₂OH, and CH₂CH₂OCH₃. R can be any amino acid side chain,including those of amino acids that are not used in proteins. Inpreferred embodiments, R is H or CH₂COO⁻ (side chain of aspartic acid).The aspartic acid side chain is useful in applications where it isdesired that the net charge of the modified Hb residue is retained. Ashas been previously determined, the retention of the charge on theGlu-43(β) residue is important for retaining a low oxygen affinity ofthe modified Hb (Rao and Acharya, 1994).

In the above formula, W is preferably NH, which creates a goodnucleophile for the PEGalation reaction. The glycinamidyl PEG thusdescribed can be produced by acetylation of PEG amine with glycine. SeeExample 2 (see also Example 1 for a method of making an analogousbifunctional glycinamidyl PEG useful as a crosslinking reagent, asdescribed below).

The CHR in the above formula can also be usefully substituted with NH,providing a PEG hydrazide. This reagent can be generated by reactingcarboxymethylated PEG with hydrazine by known methods.

For inter- or intramolecular crosslinking, the preferred formula for thenucleophilic polyalkylene glycol isH₂N—CHR—CO—W—CH₂—CH₂—[O—CH₂—CH₂]_(n)—NH—CO—W′—CHR′—NH₂ wherein n is aninteger from about 15 to about 125 where intramolecular crosslinking isdesired, and 15-250 for intermolecular crosslinking, W and W′ are eachindependently NH or O, and R and R′ are each independently an amino acidside chain. As with the preferred PEG for decoration described above, Rcan be any amino acid side chain, including those of amino acids thatare not used in proteins, but in preferred embodiments, R is H orCH₂COO—; the CHR of CHR′ can be substituted for NH; and W is preferablyNH.

The amidation reaction can be performed for about 15 min. to about 3hours, preferably for about 1 hour at room temperature. The amidationreaction may also be carried out at about 4° C. overnight if desired.The pH of the amidation reaction is preferably from about 5.8 to about7.5. In a preferred embodiment, the amidation reaction is carried out ata pH of about 7 resulting in amidation predominately at the Glu-43(β)carboxyl group of hemoglobin. The reaction can be performed in 0.1 M KClbuffer at the desired pH (6-8) using a pH-stat, to keep the pH of thereaction mixture constant with HCl, since the amidation reaction raisesthe pH (Rao and Acharya, 1994). However, it is preferred that thereaction takes place in MES buffer, preferably at about 20 mM, sincethat procedure is easier than using a pH-stat. In preferred embodiments,the molar ratio of PEG to Hb in the amidation reaction is about 50. OxyHb is the preferred conformational state of the protein since it makesthe execution of the amidation reaction protocol simpler relative tothat using deoxy Hb. See Example 3 for typical reaction conditions.

After the reaction, the Hb-polyalkylene glycol is preferably separatedfrom the unreacted reagents by molecular sieve chromatography, forexample using Sephadex® G-25 or Superose® 12, preferably in a neutralbuffer such as 10 mM phosphate buffered saline, pH 7.4. A typicalelution pattern is provided in FIG. 3.

The amidation methods described above create novel Hb-polyalkyleneglycol compositions. Thus, in some embodiments, the present inventionprovides a hemoglobin comprising a polyalkylene glycol, wherein thepolyalkylene glycol is covalently bonded to the hemoglobin with an amidelinkage at a Glu-43(β). Preferably, the polyalkylene glycol is apolypropylene glycol or a PEG, most preferably a PEG. In preferredembodiments, the Hb is HbA. It is also preferred that the Hb alsocomprises a second PEG that is covalently bonded to a Glu-22(β) with anamide linkage.

In some aspects of these embodiments, the polyalkylene glycol does notcrosslink the Hb either intramolecularly or intermolecularly.Preferably, the polyalkylene glycol has the formulaHb-(CO—NH—CHR—CO—W—CH₂—CH₂—[O—CH₂—CH₂]_(n)—R′)_(m) wherein n is aninteger from about 125 to about 500, m is an integer from 1 to 10, W isNH or O, R is an amino acid side chain, R′ is selected from the groupconsisting of OH, OCH₃, CH₂OH, CH₂OCH₃, CH₂CH₂OH, and CH₂CH₂OCH₃, andwherein at least one PEG is bonded to the Hb at Glu-43(β). In the mostpreferred embodiments, the Hb is a hemoglobin A, W is NH, n is about125, m is 6-8, R is H or CH₂COOH, and R′ is CH₂CH₂OCH₃.

In other aspects of these embodiments, the polyalkylene glycol inter- orintramolecularly crosslinks the Hb. In these aspects, theHb-polyalkylene glycol preferably has the formulaHb-CO—NH—CHR—CO—W—CH₂—CH₂—[O—CH₂—CH₂]_(n)—NH—CO—W′—CHR—NH—CO-Hb′ whereinHb and Hb′ are the same or different hemoglobin molecule, n is aninteger from about 15 to about 250, W and W′ are each independently NHor O, R is an amino acid side chain, and HbA and/or HbA′ is bonded tothe PEG with an amide linkage at Glu-43(β). In the most preferredembodiments, R is H or CH₂COOH, Hb and Hb′ are different hemoglobin Atetramers, W and W′ are both NH, and the PEG intermolecularly crosslinksHbA with HbA′.

II. Production of Hb-polyalkylene Glycols by Reductive Alkylation.

It has further been discovered that polyalkylene glycol can becovalently bonded to Hb through reductive alkylation targeted to theα-amino groups of HbA, present at Val-1(α) and Val-1(β). Like theamidation method described above, reductive alkylation has also beenpreviously used to bond small compounds to Hb (Acharya et al., 1983;Acharya and Sussman, 1984; Acharya et al., 1991). However, the utilityof applying this method to polyalkylene glycol linkage to Hb would notbe obvious to the skilled artisan because the polyalkylene glycolsutilized in the present invention are much larger than the compoundspreviously linked to Hb using reductive alkylation.

The reactivity of the amino groups of a protein for reductive alkylationis a direct correlate of their propensity to generate Schiff baseadducts with the carbonyl reagents. In the absence of any accessibilityproblems, the propensity of an amino groups to form Schiff base adductsis dictated by their pKa values and the pH of the reaction mixture,since Schiff-base adduct formation requires the unprotonated form ofamino groups. In proteins, the pKa of the α-amino groups is aroundneutral pH and is lower than those of the ε-amino groups. This featureof the α-amino groups of proteins facilitates the selective reductivealkylation of the α-amino groups of proteins around neutral pH, as faras the α-amino groups are accessible to the solvent phase for theformation Schiff base adduct and a limiting concentration of carbonylreagent (aldehyde) is used. In Hb, a tetrameric protein, there are fourα-amino groups per tetramer (for a molecular size of 64,000 Dalton) andall four α-amino groups are accessible for reductive alkylation withsimple aliphatic aldehydes. A discrimination between the α-amino groupof the α-chains [Val-1(α)] and that of the β-chains [Val-1(β)] of Hb canbe achieved by modulating the pH of the reaction medium and the amountof the carbonyl reagent present during reductive alkylation (Acharya etal., 1983). A choice of lower pH (around 6.5) and a carbonyl reagentconcentration that is just about 2 to 3 fold molar excess over that ofHb tetramer, results in targeting the reductive alkylation of Hbpredominantly to the Val-1(β) residues of Hb. With simple aliphaticaldehydes, a slightly higher excess of the reagent, about 5 fold molarexcess over the Hb tetramer, and a pH around 7.4 results in themodification of α-amino groups of Val-1(α) of Hb besides Val-1(β).Additional reductive alkylations at the ε-amino groups of Hb requiresmuch higher concentrations of the reagent (Acharya et al., 1991).

An advantage of the reductive alkylation chemistry for modification ofHb, particularly when targeted to Hb α-amino groups, is that themodification of Hb at its α-amino groups does not increase the oxygenaffinity of HbA (Acharya and Sussman, 1984, as established with lowmolecular weight neutral aldehydes). In fact, the modification of allfour α-amino groups of HbA by reductive alkylation by neutral aliphaticaldehydes results in a slight reduction in the oxygen affinity of HbA.

As adopted for linkage of polyalkylene glycols to Hb, the method of thepresent invention comprises incubating the hemoglobin with apolyalkylene glycol aldehyde and a borohydride under conditions and fora time sufficient for the polyalkylene glycol aldehyde to bond to thehemoglobin at the Val-1(β). In preferred embodiments, the polyalkyleneglycol is a PEG, the Hb is a hemoglobin A, and the borohydride is sodiumcyano borohydride (FIG. 6). Although the reaction can be performed withdeoxy Hb, the oxy form is preferred since handling Hb under oxyconditions is simpler.

The reductive alkylation reaction is preferably performed for about 15min. to about 3 hours, and more preferably for about 1 hour, at roomtemperature. The reductive alkylation reaction can also be carried outat about 4° C. overnight if desired. A preferred buffer for the reactionis 10 mM phosphate buffer, containing 150 mM NaCl. However, the use ofTris or Bis-Tris buffer increases the propensity of the reaction tooccur at Val-1(β) rather than Val-1(α). The molar ratio of polyalkyleneglycol aldehyde to Hb in the reaction is preferably from about 2:1 toabout 40:1. To achieve linkage of a greater number of polyalkyleneglycol aldehydes to the hemoglobin (i.e., as many as 6 to 8 polyalkyleneglycol aldehyde molecules) the polyalkylene glycol aldehyde:Hb molarratio should be about 20:1 to about 40:1, and, preferably, a slightlyhigher pH (8.0 to 8.5). Conversely, to achieve linkage at only one ortwo sites, i.e., the Val-1(β) residues, the reaction is carried out at apH of from about 6 to about 7, preferably at about 6.5 in Bis-Trisbuffer, and at a polyalkylene glycol aldehyde:Hb molar ratio of fromabout 2:1 to about 6:1, preferably from about 2:1 to about 3:1.

In another embodiment of this aspect of the invention, polyalkyleneglycol molecules can be attached at predominately the Val-1(α) residuesof HbA by performing the reductive alkylation method described above ata pH of from about 7 to about 8, preferably 7.4, and at a PEG to HbAmolar ratio of from about 2:1 to about 10:1, preferably 5:1. The processof attaching PEG to the Val-1(α) residues of HbA is preferably carriedout in the presence of inosine hexaphosphate (IHP), which blocks theresidues of the ββ cleft (Val-1(β)) for reaction.

In still another embodiment of this aspect of the invention,polyalkylene glycol molecules can be attached to Hb at substantially allα-amino groups and lysine residue εamino groups with low pKa values bythe reductive alkylation method described above, by performing thereaction at a pH of about 7.4 to 8.5, and at a temperature of about 37°C., and using a high molar ratio of polyalkylene glycol aldehyde:Hb(˜40:1).

After the reaction, the Hb-polyalklene glycol is preferably separatedfrom the unreacted reagents by molecular sieve chromatography, asdescribed for the results of the amidation methods.

The skilled artisan can design, without undue experimentation, a widerange of polyalkylene glycol aldehydes useful for modification of Hb byreductive alkylation. A preferred form is PEG-propionaldehyde (FIG. 7).The skilled artisan would understand that with one propionaldehydemoiety, this reagent would be useful for decoration of Hb; with twopropionaldehyde moieties (preferably at each end), the reagent would beuseful for crosslinking applications.

Another preferred polyalkylene glycol aldehyde for decoration isHOC—[CH₂]_(p)—[NH]_(q)—CH₂—CH₂—[O—CH₂—CH₂]_(n)—R wherein p is an integerfrom 2 to 6, q is 1 or 2, n is an integer from about 125 to about 500,and R is selected from the group consisting of OH, OCH₃, CH₂OH, CH₂OCH₃,CH₂CH₂OH, and CH₂CH₂OCH₃. Preferably, q is 1, n is about 125, and R isCH₂CH₂OCH₃.

For crosslinking, a preferred polyalkylene glycol aldehyde form isOHC—[CH₂]_(p)—[NH]_(—q)—CH₂—CH₂ [O—CH₂—CH₂]_(n)—[NH]_(q)—[CH₂]_(p)—CHOwherein each p is independently an integer from 2 to 6, each q isindependently 1 or 2, and n is an integer from about 15 to about 250.

The reductive alkylation methods thus described are useful for thecreation of novel Hb-polyalkylene glycol compositions. Thus, in someembodiments, the present invention provides a hemoglobin comprising apolyalkylene glycol, and wherein the polyalkylene glycol is covalentlybonded to the hemoglobin at the α-amino of a Val-1(β). Preferably, thepolyalkylene glycol is a polypropylene glycol or a PEG, most preferablya PEG. In preferred embodiments, the Hb is HbA. It is also preferredthat the Hb also comprises a second PEG that is covalently bonded to thehemoglobin at the α-amino of a Val-1(α).

In some embodiments, the polyalkylene glycol does not crosslink the Hbintramolecularly or intermolecularly. In those embodiments, a preferredHb-polyalkylene glycol composition isHbA-(NH—CH—[CH₂]_(p)—[NH]_(q)—CH₂—CH₂—[O—CH₂—CH₂]_(n)—R)_(m) wherein pis an integer from 2 to 6, q is 1 or 2, n is an integer from about 125to about 500, m is an integer from 1 to 10, and R is selected from thegroup consisting of OH, OCH₃, CH₂OH, CH₂OCH₃, CH₂CH₂OH, and CH₂CH₂OCH₃.In the most preferred embodiments, q is 1, n is about 125, m is 1-4, andR is CH₂CH₂OCH₃.

In other embodiments, the polyalkylene glycol does crosslink the Hbintramolecularly or intermolecularly. In those embodiments, a preferredHb-polyalkylene glycol composition isHb-NH—CH—[CH₂]_(p)—[NH]_(q)—CH₂—CH₂—[O—CH₂—CH₂]_(n)—[NH]_(q)—[CH₂]_(p)—CH—NH-Hb′wherein Hb and Hb′ are the same or different hemoglobin molecule, each pis independently an integer from 2 to 6, each q is 1 or 2, preferably 1,and n is an integer from about 15 to about 250. In intramolecularcrosslinking embodiments, it is preferred that Hb and Hb′ are the samehemoglobin A tetramer. In intermolecular crosslinking embodiments, it ispreferred that Hb and Hb′ are different hemoglobin A tetramers.

III. Improvements in the Production of Hb-polyalkylene Glycols byThiolation Mediated Maleimide Chemistry

An improvement has been developed for the previously discoveredpolyalkylene glycol-Hb linking method where Hb is incubated withiminothiolane and a maleidophenyl polyalkylene glycol, to form an Hbwith a polyalkylene glycol linked to a Hb thiol group through aphenylsuccinimido linkage (first disclosed in U.S. patent applicationSer. No. 425,137, which became U.S. Pat. No. 5,585,484).

As is well known, modification of the Cys-93(β) residue of hemoglobinincreases the oxygen affinity of Hb. Thus, if the oxygen affinity of thesurface decorated Hb is critical for a given therapeutic application ofacellular Hb, it may be advantageous to leave the Cys-93(β) intact. Thiscan be achieved by, for example, utilizing the amidation or reductivealkylation procedures described above. However, if one utilized theprior art to employ the thiolation-mediated maleimide method describedin U.S. Pat. No. 5,585,484, there was no way to avoid the modificationof the Cys-93(β) residue. The present invention describes an improvementin that method that results in a polyalkylene glycol-modified Hb whereinthe Cys-93(β) residue is not modified. To that end, two procedures havebeen developed for preserving the Cys-93(β) in the final product: (i)reversible blocking of the sulfhydryl group of Cys-93(β) as mixeddisulfide with glutathione and (ii) reversible blocking of thesulfhydryl group of Cys-93(β) with a dithiopyridine or a mixed disulfideof pyridine and polyalkylene glycol.

Thus, the present invention provides a method for making a hemoglobincomprising a polyalkylene glycol, wherein the polyalkylene glycol isbonded to the hemoglobin at a thiol moiety through a phenylsuccinimidolinkage. The method comprises (a) conjugating a reagent to a Cys-93(β)sulfhydryl group of the hemoglobin by a reversible method; (b) treatingthe hemoglobin with a maleidophenyl polyalkylene glycol andiminothiolane under conditions and for a time sufficient for thepolyalkylene glycol to conjugate to a hemoglobin thiol moiety through aphenylsuccinimido linkage; and (c) removing the reagent from theCys-93(β) sulfhydryl group of the hemoglobin. Step (b) is fullydescribed in U.S. Pat. No. 5,585,484.

In these methods, the reagent is either glutathione or, preferably,dithiopyridine (for example by the method described in Example 5) or amixed disulfide of pyridine and polyalkylene glycol. The pyridylpolyalkylene glycol mixed disulfide is preferred over glutathionebecause it is much easier to monitor the attachment of the largepolyalkylene glycol than the glutathione. This monitoring is preferablyperformed by molecular sieve chromatography (e.g., with a Superose® 12column), when the mixed disulfide of Hb and the polyalkylene glycol canbe detected by an acceleration in the elution of the hemoglobin due tothe increased hydrodynamic volume (a hemoglobin tetramer of 64,000molecular weight with a PEG-5000 attached behaves like a protein havinga molecular weight of 128,000). This chromatographic procedure will alsoconveniently separate the modified Hb from the unmodified Hb (ordithiopyridine) if the modification is not quantitative. By contrast,the formation of the mixed disulfide of Hb with glutathione is monitoredwith isoelectric focusing. Column chromatography must also still beperformed with the glutathione procedure to remove the unreactedglutathione. If monitoring of the reaction is not necessary, thendithiopyridine is the preferred reagent because it provides for a costeffective, convenient method.

The mixed disulfide of pyridine and polyalkylene glycol is preferably apolypropylene glycol or a PEG, more preferably a PEG. The polyalkyleneglycol of the mixed disulfide can be any convenient size, but preferablyat least about 5000 molecular weight (about 125 O—CH₂—CH₂ units for athe pyridine-PEG mixed disulfide), since the attachment of apolyalkylene glycol of that size to Hb can be easily detected as ahydrodynamic volume change.

In these methods, the maleidophenyl polyalkylene glycol is preferablyeither a 4-phenylmaleimido polyalkylene glycol or a 3-phenylmaleimidopolyalkylene glycol, most preferably a 4-phenylmaleimido polyalkyleneglycol. The polyalkylene glycol is preferably either a polypropyleneglycol or a PEG, most preferably a PEG.

For decoration, a preferred phenylmaleimido polyalkylene glycol isY—R—CH₂—CH₂—[O—CH₂—CH₂]_(n)—R′—Y′ wherein n is an integer from about 125to about 500; R is carbamate, urea, or amide; R′ is carbamate, urea,amide, or oxygen; Y is 4-phenylmaleimido or 3-phenylmaleimido; and Y′ ismethyl or hydrogen. More preferably, Y is 4-phenylmaleimido, Y′ ismethyl, R is carbamate, R′ is oxygen, and n is about 125.

For crosslinking, a preferred phenylmaleimido polyalkylene glycol isY—R—CH₂—CH₂—[O—CH₂—CH₂]_(n)—R′—Y wherein n is an integer from about 15to about 250, R and R′ are the same or different and are carbamate,urea, or amide, and Y and Y′ are the same or different and are4-phenylmaleimido or 3-phenylmaleimido. More preferably, Y and Y′ areboth 4-phenylmaleimido, and R and R′ are both carbamate.

This method thus provides novel modified hemoglobins. In someembodiments, the method provides a hemoglobin comprising a polyalkyleneglycol, wherein the polyalkylene glycol is covalently bonded to thehemoglobin at a thiol moiety through a phenylsuccinimido linkage, andwherein no polyalkylene glycol is covalently bonded to a Cys-93(β).Preferably, the polyalkylene glycol is a polypropylene glycol or apolyethylene glycol (PEG), most preferably a PEG. The hemoglobin is mostpreferably a hemoglobin A.

The polyalkylene glycol can decorate or crosslink the hemoglobin. Apreferred decorated hemoglobin has the formulaHb-(S—Y—R—CH₂—CH₂—[O—CH₂—CH₂]_(n)—R′—Y′)_(m) wherein n is an integerfrom about 125 to about 500; m is an integer from about 2 to about 16; Ris carbamate, urea, or amide; R′ is carbamate, urea, amide, or oxygen; Yis 4-phenylsuccinimido or 3-phenylsuccinimido; and Y′ is methyl orhydrogen. In more preferred embodiments, the Hb is hemoglobin A, Y is4-phenylsuccinimido, Y′ is methyl, R is carbamate, R′ is oxygen, n isabout 200, and M is an integer from about 6 to about 8.

A preferred crosslinked hemoglobin has the formulaHb-(S—Y—R—CH₂—CH₂—[O—CH₂—CH₂]_(n)—R′—Y′—S-Hb′ wherein n is an integerfrom about 20 to about 500, R and R′ are the same or different and arecarbamate, urea, or amide, Y and Y′ are the same or different and are4-phenylsuccinimido or 3-phenylsuccinimido, and Hb and Hb′ are the sameor different hemoglobin molecule. In the more preferred intramolecularlycrosslinked Hb embodiments, Hb and Hb′ are the same hemoglobin Atetramer, Y and Y′ are both 4-phenylsuccinimido, and R and R′ are bothcarbamate. In the more preferred intermolecularly crosslinked Hbembodiments, the Hb and Hb′ are different hemoglobin A tetramers, Y andY′ are both 4-phenylsuccinimido, and R and R′ are both carbamate.

The present invention also provides pharmaceutical compositionscomprising any of the compositions described above and apharmaceutically acceptable excipient. Suitable carriers include but arenot limited to various physiologically acceptable solutions known in theart such as saline solution, Ringer's solution, lactated Ringer'ssolution, Locke-Ringer's solution, Kreb's Ringer's solution, Hartmann'sbalanced saline solution, and heparinized sodium citrate acid dextrosesolution. The pharmaceutical compositions also may comprise known plasmasubstitutes and plasma expanders. The pharmaceutical compositions of thepresent invention may be used as blood substitutes, and the like, andmay be administered by conventional means including but not limited totransfusion and injection.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

EXAMPLE 1 Preparation of Bifunctional Glycinamidyl PEG

5 Grams of O,O′(2-amino propyl) polyethylene glycol 600 (diaminoPEG-600) was acylated with about two molar equivalents of BOC (tertiarybutyloxy carbonyl)Glycyl-ONSu (N-hydroxy succinmidyl ester of t-butyloxyGlycine) in dry tetrahydrofuran at room temperature. After 16 hours, theanalytical TLC showed trace amounts of unreacted amino compound. Thesolvent was evaporated under reduced pressure, and the residual oil istreated with trifluoroacetic acid:dichloromethane (TFA:DCM) at a 1:1ratio by volume. The dry product was dried under vacuum over sodiumhydroxide pellets and 4.5 grams of bifunctional glycinamidyl PEG wasrecovered.

EXAMPLE 2 Preparation of Monofunctional Glycinamidyl PEG

5 Grams of omega methoxy monoamine PEG was acylated with about one molarequivalent of N-hydroxy succinimidyl ester of t-butyloxy glycine in drytetrahydrofuran at room temperature. After 16 hours, the analytical TLCshowed trace amounts of unreacted amino compound. The solvent wasevaporated under reduced pressure, and the residual oil is treated withtrifluoroacetic acid:dichloromethane (TFA:DCM) at a 1:1 ratio by volume.The dry product was dried under vacuum over sodium hydroxide pellets and4.8 grams of monofunctional glycinamidyl PEG was recovered.

EXAMPLE 3 Preparation of hemoglobin A decorated with glycinamidylPEG-5000

A 2.0 mM solution of hemoglobin A (HbA) (in the oxy state) is dialyzedovernight at 4° C. against 20 mM MES buffer, pH 6.0. A 50 mM solution ofglycinamidyl PEG-5000, and a 20 mM solution ofN-hydroxysulfosuccinimide, both in MES buffer at pH 6.0 is alsoprepared. The HbA, glycinamidyl PEG-5000, and N-hydroxysulfosuccinimidesolutions are mixed together to final concentrations of 0.5 mM HbA, 25mM glycinamidyl PEG-5000, and 5 mM N-hydroxysulfosuccinimide. Thereaction is initiated by the addition of1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDC) from a stocksolution to a final concentration of 20 mM. The concentration of EDCstock solution is such that the volume of the solution added to initiatethe amidation reaction is less than 2% of the total reaction volume.After addition of EDC, the reaction mixture is incubated at roomtemperature for 1 hr. The PEGalated HbA is separated from anynon-PEGalated HbA by gel filtration on a Superose®-12 column with 10 mMphosphate buffered saline, pH 7.4.

EXAMPLE 4 Characterization of PEGalated Surface Decorated Hemoglobin

Amidation of HbA using 10 mM carbodiimide and glycyl ester of PEG-5000as the nucleophile (an established reaction condition to siteselectively activate the γ-carboxyl group of Glu-43(β)) generated asurface decorated product (FIG. 3) that exhibits a hydrodynamic volumecomparable to that of HbA carrying two copies of PEG-5000 chain onCys-93(β), as produced using the thiolation-mediated maleimide method ofU.S. Pat. No. 5,585,484. Therefore, the apparent molecular size of Hbsurface decorated with PEG-5000 appears to be dictated by the molecularsize of the PEG chains rather than the chemistry used to link the PEGchains to Hb.

To increase the number of PEG-chains introduced on to Hb through sidechain carboxylates, the surface decoration has been carried out in thepresence of 50 mM carbodiimide (a condition known to activate all threehigh pKa carboxyl groups of Hb). A product with much higher apparentmolecular size and very little of lower molecular size forms of HbA, wasgenerated reflecting the conjugation of the PEG at side chain carboxylgroups other than that of Glu-43(β). The apparent molecular size of thisproduct corresponded to that of the surface decorated sample of Hbgenerated by introducing 6 to 8 PEG-chains per tetramer by thethiolation mediated, maleimide chemistry based PEGalation reaction.Since both samples contain on an average six to eight PEG-5000 chainsper tetramer, the results further support the conclusion that thehydrodynamic volume of surface decorated HbA depends primarily on thenumber of PEG-5000 chains placed on the Hb tetramer and is independentof the site of attachment and the chemistry used for attaching thePEG-chains.

The molecular sieve chromatographic patterns of HbA surface decoratedwith glycine ester of PEG-5000 using 25 mM EDC, revealed that theapparent molecular size of the major product of the reaction isintermediate to that of HbA with two equivalents of glycine ethyl esterof PEG-5000 and that of the sample containing on an average six to eightequivalents of glycine ethyl ester of PEG-5000.

In an attempt to correlate the apparent increase in the molecular sizeof HbA as consequence of PEGalation, the Superose®-12 column wascalibrated by running a sample of intramolecularly crosslinked HbA (ααfumaryl HbA) intertetramerically crosslinked with bis-maleidophenylPEG-600. HbA intramolecularly crossbridged within the central cavitybetween Lys-99(α) and Lys-99(α) using dibromosalicyl fumarate (XLfumaryl αα HbA) was intermolecularly crosslinked with bis-maleidophenylPEG-600 to introduce intertetrameric crossbridged HbA. This samplecontaining intertetramerically crossbridged higher molecular weightforms of XL-fumaryl-αα HbA served as the markers for the elutionposition of multimeric forms of HbA. (FIG. 4). Each peak positioneluting earlier to the intermolecularly crossbridged HbA corresponds tothe next higher polymeric state of inter molecularly crossbridged HbA(FIG. 4). An elution position of 56 minutes on this column representsthe molecular size of tetrameric Hb, with an apparent molecular weightof 64,000. Thus, the elution position of 50 minutes on the Superose®-12column corresponds to that of octameric form with an apparent molecularweight of 128,000 daltons, the elution position around 47 minutescorrespond to that of dodecameric protein with a molecular weight of192,000 daltons, and the elution position of 44 minutes corresponds tothat of a hexadecameric species, with an apparent molecular size of256,000 daltons. The elution position of the main component of thesample generated by the amidation reaction using 50 mM EDC correspondsto that of hexadecameric (16 subunits, molecular size 256,000 Dalton)form of intra molecularly cross bridged HbA.

The progressive decrease in the retention times of HbA on amidation withthe glycine ester of PEG-5000 as the concentration of EDC (water solublecarbodimide) used in the amidation mixture increases therefore reflectsthe correlation of the number of PEG-5000 chains with the apparentincrease molecular size. 10 to 15 mM EDC reaction product predominantlycontaining two PEG-5000 chains elutes around 50 min, and representsmolecular size corresponding to that of octameric form of HbA. Theprimary product generated in the presence of 25 mM EDC elutes around 47minutes and this product corresponds to the molecular size of ahemoglobin dodecamer. The major product present in the 50 mM EDCamidated sample elutes around 44 minutes and thus corresponds closely tomolecular size of hexadecameic HbA. The correlation of the number ofPEG-5000 chains expected to be present in the major product of each ofthe amidated sample with the calculated apparent increase in themolecular size of HbA is shown in FIG. 5. There is a linear correlationbetween the increase in the hydrodynamic volume of the surface decoratedHbA and the number of PEG-chains introduced onto the protein. (FIG. 5).A mass of 10,000 dalton of PEG conjugated on to the surface of Hbincreased molecular size of Hb as much as one molecule of terameric HbA,64,000 dalton (nearly six times the actual molecular size of PEG).

The results of the above study lead us to conclude that there is alinear correlation with the number of PEG-5000 chains on the tetramerand the increase in the apparent molecular size of HbA as the moleculeis loaded up to 6 to eight PEG-5000 chains.

The possibility of decorating Hb with more than six to eight PEG-5000chains has also been attempted to establish whether this linearcorrelation is true beyond the six to eight PEG-5000 chains as well. Thethiolation mediated maleimide chemistry based PEGalation, as describedin U.S. Pat. No. 5,585,484, was used to increase the extent of thePEGalation of Hb. Though we could increase the extent of thiolationthrough this procedure, the molecular size of the product did not changeconsiderably. Thus, it appears that about six chains of PEG-5000 pertetramer represents the limiting number for surface decoration usingPEG-5000 chains for which a reasonably direct correlation exists betweenthe mass of PEG-chains introduced on to Hb and the apparent molecularsize. Though the number of thiol groups that can be introduced onto Hbincreased when the amount of the thiolating reagent (iminothiolane) wasincreased from 10-fold molar excess to 40-fold molar excess, subsequentPEGalation by maleidophenyl PEG-5000 on the Hb molecule appears to bevery inefficient because once it carries six to eight PEG-5000 chains,there appears to be resistance to the attachment of additionalPEG-chains even though the Hb has been thiolated to carry additionalthiol groups to facilitate the PEGalation. The subsequent PEGalationthus appears to proceed very slowly, and the concomitant increase in themolecular size is very limited. The elution pattern also suggests that,as the extent of thiolation increases, the product generated becomesmore and more heterogeneous. Thus a molecule of Hb with six to eightchains of PEG-5000, appears to be experiencing a ‘crowding influence’which decreases the chances of adding additional PEG-chains on to thesurface of HbA. Also, these newly added PEG-chains contribution towardan increase in the apparent molecular size appears to be very marginal.

An alternate approach of increasing the mass of PEG conjugated onto thesurface of Hb is to use PEG-chains of higher mass. For example, insteadof attaching 8 copies of maleidophenyl PEG-5000 on to the tetramer, onecould attach two copies of maleidophenyl PEG-20,000 at the twoCys-93(β). The two preparations will have nearly the same total amountof PEG per tetramer. HbA carrying two PEG-20,000 chains on Cys-93(β),was thus prepared using maleidophenyl PEG-20,000. This species of Hbwith a total PEG-mass of 40,000 dalton, elutes at a position earlier tothat of the species of Hb that carry six to eight PEG-5000 chains pertetramer (FIG. 8). As noted earlier, the latter position corresponds tothat of the hexadecameric species of HbA. Thus, the apparent molecularsize of Hb(PEG-20,000)₂ is larger than that of hexadecameric species ofHbA. We have obtained similar results with the reductive alkylation ofHb with PEG-20,000 propionaldehyde. The reaction has generated productsof Hb that exhibited a molecular sieve chromatographic patterncomparable to that of the species that carry PEG-20,000 on the twoCys-93(β). This again supports the conclusion that the apparent increasein the molecular size is dictated by the molecular mass of PEG-chainsattached to the surface of protein and not on where it is attached.

Therefore, the present study supports the conclusion that the mostefficient approach for increasing the apparent molecular size of aprotein by PEGalation is to attach lower number of longer PEG-chainsthan larger number of shorter PEG-chains.

EXAMPLE 5 Reaction of Mixed Disulfide of HbA and Dithiopyridine

HbA (0.5 mM) in phosphate buffered saline, pH 7.4 is incubated at roomtemperature with a 4 fold molar excess of dithiopyridine for two hoursand the reaction mixture is gel filtered though a column of Sephadex®G-25 equilibrated with phosphate buffered saline. The mixed disulfide ofHb with dithiopyridine is formed in nearly quantitative yields asreflected by isoelectric focusing analysis. This modified Hb is thensubjected to surface decoration with maleidophenyl PEG-5000 in thepresence of iminothiolane, under conditions where six to eight PEG-5000chains are introduced with the control Hb, two of which are present atthe two Cys-93(β) residues of HbA. The Cys-93(β) if the surfacedecorated Hb is regenerated by treating the mixed disulfide with smallmolecular weight thiols. With the mixed disulfide of pyridine and Hb,the number of PEG-chains introduced are therefore to be lower by twoPEG-5000 chains per tetramer. Nonetheless, the hydrodynamic volume ofthe product generated is not very different from that of the productobtained with the unmodified Hb. A comparison of the extent ofthiolation showed that the number of new thiols introduced into theprotein is not influenced by the formation of a mixed disulfide atCys-93(β). The results demonstrate that the elution profile of a sampleof surface decorated HbA and that of the mixed disulfide of pyridine andHbA are very similar. The number of PEG-chains present in the sample ofHbA PEGlated with a ten fold molar excess of iminothiolane are more thanenough for it to give an apparent molecular size higher than 256,000daltons. This suggests that after attaining an apparent molecular sizehigher than 256,000 daltons, the propensity of additional PEG-chains toincrease the apparent molecular size of Hb is very little. Thus, thecrowding effect suggested in Example 4 with reference to the adding morethan six to eight PEG chains a may be responsible for this observationas well.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

1. A hemoglobin comprising a polyalkylene glycol, wherein thepolyalkylene glycol is a polypropylene glycol or a polyethylene glycol(PEG), and the polyalkylene glycol is covalently bonded to thehemoglobin with an amide linkage at a Glu-43(β).
 2. The hemoglobin ofclaim 1, wherein the polyalkylene glycol is a PEG, and the hemoglobin isa hemoglobin A.
 3. The hemoglobin of claim 1, wherein the polyalkyleneglycol is a PEG, and the hemoglobin further comprises a second PEGwherein the second PEG is covalently bonded to a Glu-22(β) with an amidelinkage.
 4. The hemoglobin of claim 1, wherein the polyalkylene glycolis a PEG, and the PEG does not crosslink the hemoglobin intramolecularlyor intermolecularly.
 5. The hemoglobin of claim 4, wherein at least 6PEGs are bonded to the hemoglobin through an amide (isopeptide) linkage.6. The hemoglobin of claim 1, wherein the polyalkylene glycol is a PEG,and the PEG intramolecularly crosslinks the hemoglobin.
 7. Thehemoglobin of claim 1, wherein the polyalkylene glycol is a PEG, and thePEG intermolecularly crosslinks the hemoglobin with a second hemoglobin.8. A hemoglobin composition comprising hemoglobin (Hb) decorated withone or more PEG molecules, wherein the Hb-PEG has the formulaHb-(CO—NH—CHR—CO—W—CH₂—CH₂—[O—CH₂—CH₂]_(n)—R′)_(m) wherein n is aninteger from about 125 to about 500, m is an integer from 1 to 10, W isNH or O, R is an amino acid side chain, R′ is selected from the groupconsisting of OH, OCH₃, CH₂OH, CH₂OCH₃, CH₂CH₂OH, and CH₂CH₂OCH₃, andwherein at least one PEG is bonded to the Hb at Glu-43(β).
 9. Thehemoglobin of claim 8, wherein the Hb is a hemoglobin A, W is NH, n isabout 125, m is 6-8, R is H or CH₂COOH, and R′ is CH₂CH₂OCH₃.
 10. Ahemoglobin composition comprising at least one hemoglobin molecule (Hb),crosslinked by one or more PEG molecules, wherein the crosslinked Hb hasthe formulaHb-CO—NH—CHR—CO—W—CH₂—CH₂—[O—CH₂—CH₂]_(n)—NH—CO—W′—CHR—NH—CO-Hb′ whereinHb and Hb′ are the same or different hemoglobin molecule, n is aninteger from about 15 to about 250, W and W′ are each independently NHor O, R is an amino acid side chain, and HbA and/or HbA′ is bonded tothe PEG with an amide linkage at Glu-43(β).
 11. The hemoglobincomposition of claim 10, wherein R is H or CH₂COOH, Hb and Hb′ aredifferent hemoglobin A tetramers, W and W′ are both NH, and the PEGintermolecularly crosslinks HbA with HbA′. 12-15. (canceled)
 16. Amethod of producing a hemoglobin comprising a polyalkylene glycol,wherein the polyalkylene glycol is polypropylene glycol or polyethyleneglycol (PEG), the method comprising mixing in a suitable buffer (a) thehemoglobin, (b) a carbodiimide, and (c) a nucleophilic polyalkyleneglycol with a terminal amine having a pK_(a) below 9, and incubating themixture under conditions and for a time sufficient for the polyalkyleneglycol to covalently bond to the hemoglobin at Glu-43(β).
 17. The methodof claim 16, wherein the polyalkylene glycol is a PEG, and 6 to 8 PEGmolecules bind to the hemoglobin.
 18. The method of claim 16, whereinthe buffer is MES buffer at pH 6-8, the carbodiimide is1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide, and the mixture furthercomprises N-hydroxysulfosuccinimide.
 19. The method of claim 18, whereinthe carbodiimide is present in the mixture at about 10 mM.
 20. Themethod of claim 18, wherein the carbodiimide is present in the mixtureat about 50 mM.
 21. The method of claim 16, wherein the hemoglobin ishemoglobin A.
 22. A method of producing a hemoglobin comprising apolyethylene glycol (PEG), the method comprising mixing in a suitablebuffer (a) the hemoglobin, (b) a carbodiimide, and (c) a nucleophilicPEG with a terminal amine having a pK_(a) below 9, and incubating themixture under conditions and for a time sufficient for the PEG tocovalently bond to the hemoglobin at Glu-43(β), wherein the nucleophilicPEG has the formula H₂N—CHR—CO—W—CH₂—CH₂—[O—CH₂—CH₂]_(n)—R′ wherein n isan integer from about 125 to about 500, W is NH or O, R is an amino acidside chain, and R′ is selected from the group consisting of OH, OCH₃,CH₂OH, CH₂OCH₃, CH₂CH₂OH, and CH₂CH₂OCH₃.
 23. The method of claim 22,wherein the hemoglobin is a hemoglobin A, W is NH, n is about 125, R isH or CH₂COOH, and R′ is CH₂CH₂OCH₃.
 24. The method of claim 16, whereinthe nucleophilic PEG has the formulaH₂N—CHR—CO—W—CH₂—CH₂—[O—CH₂—CH₂]_(n)—NH—CO—W′—CHR′—NH₂ wherein n is aninteger from about 15 to about 250, W and W′ are each independently NHor O, and R and R′ are each independently an amino acid side chain. 25.The hemoglobin composition of claim 24, wherein W and W′ are both NH,and R or R′ is each independently H or CH₂COOH. 26-79. (canceled)